Squamous cell differentiation, an important tissue remodeling process, has been implicated in many cellular events from promoting barrier function to rendering cell transformation and carcinogenesis in the airway. Vitamin A deficiency is one of known causes for airway squamous cell differentiation. We undertook a study to determine whether Rad, whose expression is induced by vitamin A deficiency, modulates airway squamous cell differentiation, such as the expression of a small proline-rich protein, SPRR1B, a hallmark of squamous cell differentiation in human tracheobronchial epithelial (TBE) cells. Immunohistochemical studies of human tracheobronchial and esophageal tissues indicated that Rad protein is significant presence in ciliated and squamous cells, but scarce in mucous cells. Both Northern blot and Real Time RT-PCR analysis demonstrated that the expression of Rad, a member of Rad/Gern/Kir (RGK) Ras-like subfamily, is temporally and spatially correlated with the induction of SPRR1B, but inversely correlated with the level of MUC5B/MUC5AC expression in human TBE cells. Utilizing Tet-on Rad and mutant Rad(S66N) inducible cell systems in HBE1 cells, we demonstrated that Rad, but not the mutant Rad(S66N) expression is a crucial signaling molecule involved in coordinating airway epithelial cell differentiation between the squamous and mucous cell phenotypes. Initial studies suggested that Rad through the interaction with microtubule exerts a protein kinase C (PKC)-mediated signaling pathway in the induction of SPRR1B expression. A similar PKC-mediated signaling is apparently involved in the attenuation of MUC5B/MUC5AC expression by Rad. These preliminary results strongly suggest that Rad may serve as a "switch" factor that is able to programming airway epithelial cell differentiation between squamous and mucous cell phenotypes. In this proposal, we hypothesize that Rad protein regulates airway epithelial call differentiation through a protein kinase C (PKC)/MAPK/AP-1 pathways. In order to test the hypothesis, Aim 1 is to determine the structure-functional relationship of Rad to interact with microtubule and the consequence of the interaction in the regulation of SPRR1B expression. Aim 2 is to see how and which PKC isozyme is activated by Rad-microtubule complex. Aim 3 is to elucidate down stream signaling after Rad-microtubule/ PKC activation for SPRR1B expression. Aim 4 is to elucidate the molecular nature of the transcriptional activation of SPRR1B expression by Rad. We will elucidate both cis-elements and trans-acting factors involved in the process. Aim 5 is to develop a biological paradigm for primary human airway epithelial cells/tissues of the function of Rad in the attenuation of airway epithelial cell differentiation. These to-be-obtained results will not only be of interest to general biological science, but also assist us to develop a potential effective therapeutic tool for the control of the plasticity of epithelial cells that are frequently seen in airways under various physiological and pathological stages. In summary, we have proposed a comprehensive approach utilizing combined cell line, primary cells, and ex vivo tissues to test this hypothesis. [unreadable] [unreadable]